Differential regulation of chemokine expression by Th1 and Th2 cytokines and mechanisms of eotaxin/CCL-11 expression in human airway smooth muscle cells

Abstract

Background: Airway smooth muscle (ASM) cells may contribute to the pathogenesis of asthma including airway inflammation and remodeling. We focused our study on the regulation of chemokine expression by cytokines and analyzed the mechanisms of eotaxin/CCL-11 expression in ASM cells.

Methods: Human ASM cells were cultured in vitro and treated with IL-4, interferon-gamma (IFNgamma), and tumor necrosis factor-alpha (TNFalpha). Secretion of chemokines into the culture medium was analyzed by ELISA. Expression of eotaxin mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Binding of transcription factor signal transducer activator of transcription (STAT) 6 to the eotaxin promoter-derived DNA was analyzed by pull-down Western blot. To assess transcriptional regulation of eotaxin, cells were transfected with eotaxin promoter-luciferase reporter plasmids, and activity was determined by dual luciferase assay.

Results: The Th2 cytokine IL-4 preferentially stimulated the expression of the CC chemokine receptor (CCR) 3-ligand chemokines eotaxin, eotaxin-3, and MCP-4. The Th1 cytokine IFNgamma stimulated the expression of chemokines IP-10 and RANTES. IL-4 stimulated nuclear translocation of signal transducer activator of transcription 6 (STAT6) and its binding to the eotaxin promoter region. IL-4 activated the eotaxin promoter and its activity was inhibited by mutation of the binding site for STAT6 in the promoter.

Conclusions: The Th2 cytokine IL-4 preferentially stimulated the expression of CCR3 ligand chemokines including eotaxin in ASM cells. The transcription factor STAT6 may play a pivotal role in the activation of eotaxin transcription in response to IL-4.

Fig. 1

Secretion of chemokine proteins into the medium of airway smooth muscle (ASM) cells. a Eotaxin/CCL11. b Eotaxin-3/CCL26. c MCP-4/CCL13. d RANTES/CCL5. e IP-10/CXCL10. Medium was collected from cells stimulated with 10 ng/ml IL-4, IFNγ, and/or TNFα for 24 h and subjected to ELISA. The data are presented as the mean ± SEM of five independent experiments. * p < 0.05 compared to nonstimulated control cells. ** p < 0.05 compared to stimulation with IL-4. *** p < 0.05 compared to stimulation with TNFα or IFNγ.

Fig. 2

Regulation of eotaxin mRNA expression in airway smooth muscle (ASM) cells. RNA was extracted from cells which had been stimulated with 10 ng/ml IL-4, and/or TNFα for 24 h and subjected to real-time PCR. Levels of mRNA were calculated as fold induction compared with nonstimulated control. The data are presented as the mean ± SEM of three independent experiments. * p < 0.05 as compared with nonstimulated cells. ** p < 0.05 as compared with the cells stimulated with IL-4.

Fig. 3

Nuclear translocation of STAT6 and its binding to the proximal region of the eotaxin promoter. Nuclear protein was extracted from airway smooth muscle (ASM) cells treated with or without 10 ng/ml IL-4, and/or TNFα for 30 min. The extract was precipitated with an oligonucleotide probe which contains a putative binding site for STAT6 derived from the eotaxin promoter. The extract was analyzed by Western blot using an STAT6 Ab. Results are representative of three independent experiments.

Fig. 4

Luciferase reporter assays of the eotaxin promoter in airway smooth muscle (ASM) cells. Cells were transfected with the eotaxin promoter-luciferase reporter plasmids illustrated in the figure (pEotx.1363 and M1) and control vector pRL-TK. Forty-eight hours later, cells were stimulated for 20 h with or without 10 ng/ml IL-4 and TNFα. The relative luciferase activity was calculated as fold induction compared with the control value. The data are presented as the mean ± SEM of a total of five independent experiments. * p < 0.05 as compared with non-stimulated control cells. a pEotx.1363. b pEotx. M1.